The specific aims of this proposal test the central hypothesis that expression profiles of cellular glycans can serve as unique and sensitive fingerprints, capable of defining specific cell types or reporting cell status during culture. The development of markers and reagents for purifying specific sub-populations of cells is a high priority for advancing the therapeutic and investigational uses of hESCs. Despite their predominant localization to the cell surface, glycoconjugates have been under targeted for such uses. This deficiency reflects the lack of broadly accessible techniques or expertise for characterizing these complex macromolecules. We have developed a sophisticated suite of tools for quantitatively analyzing, with unprecedented depth, the glycans, glycoproteins, and glycolipids isolated from small amounts of material. We have successfully applied these tools to assess glycan expression changes associated with the differentiation of mouse ESCs and now propose to apply our developed glycomic and glycoproteomic techniques to questions of fundamental importance for human ESC biology. Our tools will allow us to screen for changes in glycosylation that occur globally or on specific molecules as a result of aneuploidy, cell culture conditions, passage number, cell pedigree, and differentiation. In Specific Aim 1, we will define the glycomic fingerprints of hESC lines, and hESCs differentiated toward definitive endoderm, mesoderm, and neural precursor populations. The impact of aneuploidy and cell culture conditions will also be assessed. In Specific Aim 2, we will map identified glycan markers, which define hESCs or differentiated cell fates, to specific sites on proteins and lipid cores. In Specific Aim 3, we will generate tools for detecting and enriching sub-populations of hESCs and differentiated cells based on the expression of specific glycomic and/or glycoproteomic markers. Completion of these aims will not only provide novel approaches for characterizing, defining, and enriching specific cell types but will also provide a basis for exploring the role of glycosylation in hESC self renewal and differentiation.